The enzyme cyclodextrin glucosyl transferase (CGTase; E.C 2.4.1.19) has been identified in a limited group of bacteria, and catalyzes the partial conversion of starch, amylose, starch and amylose derivatives, glycogen, partially hydrolyzed starch, or maltooligosaccharides to cyclic oligosaccharides known as cyclodextrins The three most common forms of cyclodextrins are designated as alpha, beta, and gamma, being molecular rings containing 6, 7, and 8 glucopyranose units respectively Cyclodextrins find application as encapsulating agents and as additives for improving the handling and the functional properties of food, agricultural, and pharmaceutical products.
Cyclodextrin glucosyltransferase has been used almost exclusively in batch reactions. Very little information has been published about the preparation and use of cyclodextrin glucosyltransferases in immobilized form. Nakamura and Horikoshi describe in Biotech. and Bioeng. 19, 87-99 (1977) the adsorption of CGTase, chemically modified by succinylation, on a vinylpyridine copolymer. The enzyme is bound solely through ionic forces, and has the disadvantage of being gradually desorbed from the support during continuous operation. Kato and Horikoshi describe in Biotech. and Bioeng. 26, 595-598 (1984) the adsorption of a native CGTase to DIAION HP-20 resin; however, the authors report that only about 11% of the initial enzymatic activity was retained as a result of the immobilization, and as in the previous example, the enzyme desorbed from the resin during continuous operation. Ivony et al [Journal of Applied Biochemistry 5, 158-164 (1983)] and Boross et al (U.S. Pat. No. 4,593,004) describe a method for the covalent attachment of CGTase to a support using carbodiimide chemistry. This method prevents the leakage of CGTase from the support. However, the reported retention of activity as a result of the immobilization was only 3.4-6.3%; in addition, carbodiimide reagents can give rise to deleterious chemical modifications of enzymes and are relatively expensive coupling agents for use in an industrial process. Furthermore, the measured thermal stability of the CGTase immobilized by this method was low, showing a half-life of only about 63 minutes at 50.degree. C. Clearly, methods for producing an immobilized CGTase enzyme which does not desorb, is stable to operational conditions, and has a high catalytic activity after immobilization are desired